EP2784149A1 - Verfahren und Vorrichtung zur Messung von Kontraktionseigenschaften von künstlich hergestellten Herzgewebe-Konstrukten - Google Patents

Verfahren und Vorrichtung zur Messung von Kontraktionseigenschaften von künstlich hergestellten Herzgewebe-Konstrukten Download PDF

Info

Publication number
EP2784149A1
EP2784149A1 EP13160816.8A EP13160816A EP2784149A1 EP 2784149 A1 EP2784149 A1 EP 2784149A1 EP 13160816 A EP13160816 A EP 13160816A EP 2784149 A1 EP2784149 A1 EP 2784149A1
Authority
EP
European Patent Office
Prior art keywords
support
heart tissue
support element
engineered heart
tissue construct
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP13160816.8A
Other languages
English (en)
French (fr)
Inventor
Thomas Eschenhagen
Ingra Vollert
Jörg Müller
Christoph Warncke
Jördis Weiser
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Technische Universitaet Hamburg TUHH
Universitatsklinikum Hamburg Eppendorf
Tutech Innovation GmbH
Original Assignee
Technische Universitaet Hamburg TUHH
Universitatsklinikum Hamburg Eppendorf
Tutech Innovation GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Technische Universitaet Hamburg TUHH, Universitatsklinikum Hamburg Eppendorf, Tutech Innovation GmbH filed Critical Technische Universitaet Hamburg TUHH
Priority to EP13160816.8A priority Critical patent/EP2784149A1/de
Priority to ES14722997T priority patent/ES2881848T3/es
Priority to PCT/EP2014/055981 priority patent/WO2014154704A1/en
Priority to EP14722997.5A priority patent/EP2978835B1/de
Priority to US14/780,199 priority patent/US10172712B2/en
Publication of EP2784149A1 publication Critical patent/EP2784149A1/de
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/24—Heart valves ; Vascular valves, e.g. venous valves; Heart implants, e.g. passive devices for improving the function of the native valve or the heart muscle; Transmyocardial revascularisation [TMR] devices; Valves implantable in the body
    • A61F2/2472—Devices for testing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/103—Detecting, measuring or recording devices for testing the shape, pattern, colour, size or movement of the body or parts thereof, for diagnostic purposes
    • A61B5/11—Measuring movement of the entire body or parts thereof, e.g. head or hand tremor, mobility of a limb
    • A61B5/1107—Measuring contraction of parts of the body, e.g. organ, muscle
    • A61B5/1108—Measuring contraction of parts of the body, e.g. organ, muscle of excised organs, e.g. muscle preparations
    • G—PHYSICS
    • G09—EDUCATION; CRYPTOGRAPHY; DISPLAY; ADVERTISING; SEALS
    • G09B—EDUCATIONAL OR DEMONSTRATION APPLIANCES; APPLIANCES FOR TEACHING, OR COMMUNICATING WITH, THE BLIND, DEAF OR MUTE; MODELS; PLANETARIA; GLOBES; MAPS; DIAGRAMS
    • G09B23/00—Models for scientific, medical, or mathematical purposes, e.g. full-sized devices for demonstration purposes
    • G09B23/28—Models for scientific, medical, or mathematical purposes, e.g. full-sized devices for demonstration purposes for medicine
    • G09B23/30—Anatomical models
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
    • A61B2562/02—Details of sensors specially adapted for in-vivo measurements
    • A61B2562/0252—Load cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
    • A61B2562/02—Details of sensors specially adapted for in-vivo measurements
    • A61B2562/0261—Strain gauges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
    • A61B2562/02—Details of sensors specially adapted for in-vivo measurements
    • A61B2562/028—Microscale sensors, e.g. electromechanical sensors [MEMS]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2240/00—Manufacturing or designing of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
    • A61F2240/001—Designing or manufacturing processes
    • A61F2240/008—Means for testing implantable prostheses

Definitions

  • the invention is directed to a novel method for measuring contraction characteristics of engineered heart tissue constructs which is based on the mechanical coupling of the construct to a support element which comprises or is mechanically coupled to a piezoelectric element.
  • An apparatus for carrying out the method of the invention is also provided.
  • Myocardial infarction and heart failure represent the main cause of death in industrialized countries.
  • the loss of terminally differentiated cardiac myocytes which is associated with these pathologies accounts for a decrease in myocardial function which can lead to total organ failure or trigger compensatory mechanisms like hypertrophy of the remaining myocardium and activation of neurohumoral systems.
  • EHT engineered heart tissue
  • engineered heart tissue constructs may serve as in vitro models for predictive toxicology and target validation.
  • EHT-based models take advantage of a particularly physiological cellular environment.
  • cardiac tissue engineering approaches include the use of solid, preformed scaffolds ( Carrier et al. (1999), Biotechnol Bioeng., 64:580-589 ; Engelmayr et al. (2008), Nat Mater., 7:1003-1010 ; Li et al. (2000), J Thorac Cardiovasc Surg., 119:368-375 ; Leor et al.
  • a technique that has attracted considerable attention in the past is the generation of constructs in preformed casting molds using hydrogels such as collagen I, Matrigel, fibronectin, or fibrin ( Eschenhagen et al. (1997), FASEB J, 11:683-694 ; Zimmermann et al. (2002), Circ Res., 90:223-230 , Naito et al. (2006), Circulation, 114 (1 Suppl):I-72-I-78 ; Huang et al. (2007), J Biomed Mater Res A, 80:719-731 ; Bian et al. (2009), Nat Protoc., 4:1522-1534 ).
  • hydrogels such as collagen I, Matrigel, fibronectin, or fibrin
  • EHTs need to be engineered with contractile features that lend significant support to the failing heart tissue in the patient. For this reason, the ability to precisely assess the contraction characteristics of an EHT construct is of utmost importance in preparation of tissue implantation into a patient.
  • EHTs drug screening platforms and toxicology models that make use of EHTs are based on monitoring the change in the contraction characteristics of the tissue constructs upon contact with potential drugs and toxic compounds. Thus, it is a prerequisite that the contractile force generated by an EHT can be reliably determined in such screening platform or toxicology model.
  • optical surveillance systems The second commonly used approach for determining EHTs contraction involves optical surveillance systems ( Hansen et al. (2010), Circ. Res., 107(1):35-44 ). Compared to organ bath measurements, optical surveillance systems can be considered as an improvement. However, this approach still does not allow the simultaneous analysis of a high number of EHTs and is therefore unsuitable for multiplex testing. This is because a video camera can only record a single EHT at a defined point in time. Another disadvantage resides in the generation of a high volume of data. More importantly, however, the evaluation of the contractile force in optical surveillance systems is indirectly achieved measuring the deflection of elastic holdings.
  • optical surveillance systems require an auxotonic muscle contraction, while the generation of contractile force in heart tissue is normally measured isometrically, i.e. force generation without shortening of the muscle. Therefore, it is an object of the present invention to provide a method that allows the direct and continuous measurement of contractile characteristics of EHT constructs of different size and geometry.
  • the method should be cost-effective and less laborious than the presently used methods. Ideally, the method should also be amenable to automation and multiplex testing.
  • a method for measuring contraction characteristics of engineered heart tissue constructs comprises providing one or more engineered heart tissue constructs and attaching each of these engineered heart tissue constructs to a separate first support element, e.g. by mechanically coupling, mounting, adhering or otherwise connecting the respective engineered heart tissue construct to the respective first support element.
  • first support element e.g. by mechanically coupling, mounting, adhering or otherwise connecting the respective engineered heart tissue construct to the respective first support element.
  • engineered heart tissues are three dimensional, contractile, force-generating tissue structures.
  • the engineered heart tissue constructs which shall be analyzed in terms of their contraction characteristics, can be prepared in accordance with methods and protocols which are known in the art.
  • the engineered heart tissue constructs for use in the method of the present invention have been prepared in pre-formed casting molds using solubilised scaffold material, such as collagen I, fibronectin or fibrin.
  • solubilised scaffold material such as collagen I, fibronectin or fibrin.
  • PLoS One, 6(10):e26397 disclose the preparation of strip-shaped engineered heart tissue constructs from human embryonic stem cells.
  • Stem cells are first differentiated to cardiac myocytes by culturing them in the presence of a mixture of growth factors. After enzymatic dissociation, the cells are mixed with fibrin and thrombin and poured into slit-formed agarose casting molds in which two elastic silicone posts were inserted from above. After fibrin polymerisation, the cells were maintained under standard cell culture condition until the onset of contraction.
  • the engineered heart tissue constructs for use in the method of the present invention can be prepared in accordance with one of the above methods. After the constructs have formed, they can be manually transferred to an apparatus as described below for measuring their contraction characteristics. For this purpose, engineered heart tissue constructs that have been isolated from cell culture medium are attached to the separate first support element.
  • the engineered heart tissue constructs are grown in place on the support elements.
  • providing an engineered heart tissue construct and attaching it to a support element occurs generally at the same time.
  • the step of providing one or more engineered heart tissue constructs and the step of attaching each of these engineered heart tissue constructs to a separate first support element may in fact constitute a single step carried out simultaneously.
  • the engineered heart tissue constructs are preferably prepared using casting molds which are arranged such that the tissue construct is grown directly in contact with the first support element.
  • a strip-shaped casting mold can be positioned such that the first support element and, optionally, the associated second support element extend downwardly into the casting mold.
  • a liquid reconstitution mixture is poured into the casting mold such that the one or more support elements are immersed into the liquid.
  • an engineered heart tissue construct is formed around the one or more support elements.
  • Each such first support element to which an engineered heart tissue construct is attached or coupled comprises or is mechanically coupled to at least one piezoelectric element which is the sensing element of a piezoelectric transducer.
  • the piezoelectric element is a part of the first support element or is provided as a separate element mechanically coupled thereto in such a manner that the below functions can be fulfilled.
  • the piezoelectric element is a part of the first support element, and in that case it is even possible that the first support element is constituted by the piezoelectric element.
  • the first support element also comprises other parts or elements, e.g. in order to facilitate coupling to an engineered heart tissue construct, to protect the piezoelectric element from environmental influences or for mechanical or stability reasons.
  • the attachment, such as the mechanical coupling, of each of the one or more engineered heart tissue constructs to its associated first support element is effected in such a manner that upon contraction - and relaxation - of the respective engineered heart tissue construct a load or force is transferred to the piezoelectric element of the respective first support element and the respective piezoelectric transducer generates and outputs a corresponding electrical sensor signal characteristic of the load or force and/or of a change of the load or force applied to the respective first support element by the respective engineered heart tissue construct due to the contraction - and preferably also expansion - thereof.
  • the piezoelectric element is chosen and arranged suitably on or in the first support element, the attachment, such as the mechanical coupling, is established at a suitable location, and the engineered heart tissue construct is oriented suitably, such that upon contraction - and expansion - a load or force transfer takes place which induces a corresponding sensor signal indicative of the contraction - and expansion. Consequently, the type and arrangement of the piezoelectric element and the attachment of an engineered heart tissue construct must be adapted to each other. Generally, piezoelectric elements respond to compressive loads. In this connection it should be noted that it is generally difficult or at least more difficult to measure static loads or forces with piezoelectric elements and transducers.
  • the corresponding sensor signal generated by the respective piezoelectric transducer is analyzed, and at least one contraction characteristic of the engineered heart tissue construct is derived on the basis of the analysis.
  • Contraction characteristics which can be derived from the sensor signals include, e.g., contraction force, contraction frequency, contraction velocity, relaxation velocity, contraction time, relaxation time.
  • This method has the advantage that the amount of data produced is significantly smaller than the amount of data produced by prior art methods. Therefore, the data analysis and the determination of contraction characteristics is greatly facilitated and can be carried out in less time and with less hardware and work costs.
  • the method allows for easily measuring in real time and continuously multiple engineered heart tissue constructs at the same time and over extended periods of time of up to several months. Further, it is advantageously possible to measure engineered heart tissue constructs of different geometries, such as, e.g., strip-shaped, ring-shaped or patch-shaped.
  • the attachment, e.g. the mechanical coupling, of each of one or more engineered heart tissue construct to a separate first support element comprises, for each of the engineered heart tissue constructs, attaching the respective engineered heart tissue construct to one, two, three, four or more second support elements.
  • attachment may be effected e.g. by mechanically coupling, mounting, adhering or otherwise connecting the respective engineered heart tissue construct to the respective second support element or by growing the respective engineered heart tissue construct in place onto the respective second support element.
  • Each such second support element is spaced - preferably in its entirety, but at least with a portion thereof - from the associated first support element.
  • the space between said at least one first support element and said at least one second support element is between 5 and 50 mm, more preferably between 10 and 25 mm, so that the engineered heart tissue which is formed between these elements has a length in this range. It is particularly preferred that the space between said at least one first support element and said at least one second support element is between 10 and 12 mm.
  • each second support element engages the engineered heart tissue construct at a location spaced from the location at which the associated first support element engages the engineered heart tissue construct.
  • the engineered heart tissue construct is then supported between the first support element and the associated one or more second support elements, possibly in a stretched state.
  • each of the first support elements is attached to and extends from a base element, which may be provided, e.g., in the form of a frame and/or plate and may be constituted by a printed circuit board.
  • a base element is then arranged or oriented such that each of the first support elements extends downwardly from the base element while carrying out the measurement.
  • this advantageously allows for disposing the engineered heart tissue construct (s) in a container or well while the base element is supported at an edge or surrounding region of the opening of the container or well.
  • each of the second support elements is attached to and extends from the base element such that, when said base element is arranged such that each of the first support elements extends downwardly from the base element, each of the second support elements likewise extends downwardly from the base element. In this orientation each of the engineered heart tissue constructs is suspended on and between the associated first and second support elements.
  • any embodiment comprising arranging the base element such that the one or more first support elements and - if present - the one or more second support elements extend downwardly therefrom, it is advantageous to arrange the base element on the surface of a suitably dimensioned multi-well plate such that each of the first support elements together with - if present - its associated second support element(s) extends downwardly from the base element in a different one of the wells of the multi-well plate.
  • the engineered heart tissue constructs are attached, e.g. mechanically coupled, to the first and second support elements such that each is arranged in a different one of the wells. It is then possible to carry out the measurements in culture media, in order to maintain the engineered heart tissue constructs functional, or in different selected media in order to study their impact on the contraction characteristics.
  • each of the first support elements is preferably rigid, and then, for example, the following measurements can be carried out:
  • each such first support element comprises or is mechanically coupled to at least one piezoelectric element which is the sensing element of a piezoelectric transducer, i.e. the piezoelectric element is a part of the first support element or is provided as a separate element mechanically coupled thereto in such a manner that the functions mentioned below and above can be fulfilled.
  • the piezoelectric element is a part of the first support element, and in that case it is even possible that the first support element is constituted by the piezoelectric element.
  • the first support element also comprises other parts or elements, e.g. in order to facilitate coupling to an engineered heart tissue construct, to protect the piezoelectric element from environmental influences or for mechanical or stability reasons.
  • each of the first support elements comprises a support portion adapted for attaching an engineered heart tissue construct thereto.
  • the support portion may simply be constituted by the entire first support element or by an arbitrary portion of the first support element.
  • the first support elements comprises a predetermined portion as support portion, which may be, e.g., marked or specifically constructed for facilitating attachment, so that in operation engineered heart tissue constructs can be attached to the first support elements at defined locations thereof.
  • the type and arrangement of the piezoelectric element with respect to the respective first support element and the location and configuration of the support portion are chosen such that after attaching an engineered heart tissue construct to the support portion the respective engineered heart tissue construct can be arranged and stabilized in such a manner that upon contraction thereof a load or force is transferred to the respective piezoelectric element and the respective piezoelectric transducer generates and outputs a corresponding electrical sensor signal characteristic of the load or force applied to the respective first support element by the respective engineered heart tissue construct due to the contraction thereof.
  • the apparatus further comprises, for each of the one or more first support elements, one or more second support elements attached to and extending from the base element.
  • Each such second support element comprises a support portion adapted for attaching an engineered heart tissue construct thereto.
  • the support portion may simply be constituted by the entire second support element or by an arbitrary portion of the second support element.
  • the second support elements comprises a predetermined portion as support portion, which may be, e.g., marked or specifically constructed for facilitating attachment, so that in operation engineered heart tissue constructs can be attached to the second support elements at defined locations thereof.
  • each such support portion of each such second support element is spaced from and arranged with respect to the support portion of the respective first support element, such that, after attaching an engineered heart tissue construct to the support portion of one of the first support elements and to the support portion of each respective associated second support element, the respective engineered heart tissue construct is engaged by the support portion of the respective first support element at a location spaced from the location at which the support portion of each of the respective associated second support elements engage the engineered heart tissue construct, so that it is supported, preferably in a stretched state, between the respective first support element and the respective second support elements.
  • each of the second support elements is entirely spaced from the associated first support element.
  • each of the first support elements and each of the second support elements extend from a same side of the base element, and that the base element is adapted to be disposed on the surface of a suitably dimensioned multi-well plate, such that each of the first support elements extends, together with its associated second support elements, into a separate one of the wells of the multi-well plate.
  • the support portions of the first and second support elements are then arranged and located such that each engineered heart tissue construct attached to the support portions of one of the first support elements and the associated second support elements is suspended on and between the respective first and second support elements and disposed inside the respective one of the wells of the multi-well plate.
  • each of the first support elements is rigid, and that
  • the corresponding second support elements each comprise a flexible elongate tubular element - e.g. made of silicone - and a rigid elongate element shaped and dimensioned to be selectively inserted into and removed from the tubular element for selectively configuring the respective second support element to be flexible and rigid. This is a particularly simple and reliable construction of such second support elements.
  • the support portions of the first and - if present - second support elements comprise or are made of silicone.
  • such support portions may also comprise or be made of other flexible, bio-compatible materials.
  • the support portions may comprise or be a region of increased surface roughness as compared to the remainder of the respective support element.
  • each of the first support elements comprises an elongate section extending from the base element, and the respective support portion is preferably located at or near the longitudinal end of the elongate section remote from the base element.
  • the support portion comprises or is a portion extending transversely and, more preferably, perpendicularly with respect to the elongate section to opposite sides of the elongate section.
  • the support portion in addition to such a portion extending transversely or perpendicularly with respect to the elongate section to opposite sides thereof the support portion preferably also comprises a portion of the elongate section immediately adjacent to the transversely or perpendicularly extending portion.
  • an engineered heart tissue construct may then advantageously be looped or grown around the elongate section adjacent to the transversely or perpendicularly extending portion (and preferably also around the transversely or perpendicularly extending portion) such that the transversely or perpendicularly extending portion prevents the engineered heart tissue construct from slipping from the elongate section and the respective support element. If the engineered heart tissue construct adheres to the elongate section, the transversely or perpendicularly extending portion constitutes an additional securing means.
  • the elongate section which is preferably straight, may be, e.g., bar- or strip-shaped.
  • the transversely or perpendicularly extending portion of the support portion may be advantageously, e.g., disk-, plate-, dish- or bar-shaped.
  • a disk-, plate- or dish-shaped transversely or perpendicularly extending portion resulting in, e.g., a generally mushroom-shaped configuration of the first support element with the foot of the "mushroom" being connected to the base element, may be used for relatively weak engineered heart tissue constructs, whereas a bar-shaped portion extending transversely or perpendicularly with respect to the elongate section may be used for relatively strong engineered heart tissue constructs.
  • the first support element may, in particular, be chosen to have a T-shaped configuration, with the base of the "T" being connected to the base element.
  • each of the second support elements comprises an elongate section extending from the base element, and the respective support portion is preferably located at or near the longitudinal end of the elongate section remote from the base element. Also in this case it is particularly preferred if the support portion comprises or is a portion extending transversely and, more preferably, perpendicularly with respect to the elongate section to opposite sides of the elongate section.
  • the support portion in addition to such a portion extending transversely or perpendicularly with respect to the elongate section to opposite sides thereof the support portion preferably also comprises a portion of the elongate section immediately adjacent to the transversely or perpendicularly extending portion.
  • an engineered heart tissue construct may then advantageously be looped or grown around the elongate section adjacent to the transversely or perpendicularly extending portion (and preferably also around the transversely or perpendicularly extending portion) such that the transversely or perpendicularly extending portion prevents the engineered heart tissue construct from slipping from the elongate section and the respective support element. If the engineered heart tissue construct adheres to the elongate section, the transversely or perpendicularly extending portion constitutes an additional securing means.
  • the elongate section which is preferably straight, may be, e.g., bar- or strip-shaped.
  • the transversely or perpendicularly extending portion of the support portion may be advantageously, e.g., bar-, disk-, dish- or plate-shaped. If it extends transversely or perpendicularly with respect to the elongate section, the second support element may, in particular, be chosen to have a T-shaped or mushroom-shaped configuration, with the foot of the "T" or “mushroom” being connected to the base element. In the case of a strong engineered heart tissue construct choosing a T-shaped configuration with a support portion being or comprising a bar-shaped portion may be suitable. However, in the case of the second support elements it is preferred to account for the strength of the engineered heart tissue construct by using more or less second support elements.
  • each of the piezoelectric transducers is electrically connected to electronic circuitry integrated into or disposed on the base element and adapted for conditioning, converting, processing and/or analyzing the respective sensor signals.
  • a single electronic component or circuit may be electrically connected to all piezoelectric transducers, or separate electronic circuitry, which may or may not be interconnected, may be electrically connected to different ones of the piezoelectric transducers.
  • the base elements is or comprises a printed circuit board on which the circuitry is provided and to which the first and - if present - second support elements are attached. Providing the electronic circuitry on the base element ensures short signal paths and, consequently, fast response times and reduced noise and interference.
  • one or more or all of the first support elements comprise at least one strain gauge mechanically coupled to the respective first support element, such that after attaching an engineered heart tissue construct to the support portion of the respective first support element the engineered heart tissue construct can be arranged such that upon contraction thereof a load is transferred to the respective at least one strain gauge and the respective at least one strain gauge generates and outputs a corresponding electrical strain gauge signal characteristic of the static load applied by the respective engineered heart tissue construct to the respective first support element due to the contraction thereof.
  • Such strain gauges may likewise be coupled to electronic circuitry of the type and arrangement mentioned above. Although strain gauges have a rather low sensitivity as compared to the piezoelectric transducers, they can be advantageously combined therewith in order to also provide for measurements of static force characteristics of the engineered heart tissue constructs.
  • the apparatus 1 shown in Figure 1 comprises a planar plate-shaped base element 2 having two generally rectangular cut-outs 3.
  • upper surface 4 of the base element 2 electronic circuitry comprising an integrated circuit component 5 as well as a flexible strip 6 including electrical conduits are mounted.
  • the component 5 is located between the two cut-outs 3.
  • the component 5 is coupled to different piezoelectric transducers in order to process their signals, and is also coupled to the electrical conduits of the strip 6 in order to provide the processed signals to exterior means.
  • the base element 2 is a printed circuit board and signal conduction to and from the component 5 is effected by means of conductive traces (not shown) of the printed circuit board 2.
  • a projection 7 extends into the cut-out 3 in the same plane with the remainder of the base element 2.
  • a straight, elongate support element 8, 9 is attached such that the support elements 8, 9 extend downwardly for the, in Figure 1 , lower surface of the base element 2 in a manner perpendicularly to the plane defined by the base element 2.
  • one of the projections 7 carries a support element 8 and the other projection 7 carries a support element 9, so that overall two support elements 8 of identical construction and two support elements 9 of identical construction are present.
  • the two support elements 8 are attached to the projections 7 closest to the component 5.
  • Each support element 8 comprises two strip shaped piezoelectric elements 10 sandwiching between them an intermediate layer 18 stabilizing the piezoelectric elements 10 and possibly providing support for electrical conduits and/or circuitry operatively coupled to the piezoelectric elements 10.
  • This sandwich structure forms an elongate section 11, to the longitudinal end of which remote from the respective projection 7 a dish-shaped silicone element 12 having a circular shape and extending perpendicularly to the direction of extension of the elongate section 11 is attached.
  • the element 12 has rounded edges.
  • the entire elongate section 11 or at least the piezoelectric elements 10 may be provided with a protective coating (not shown).
  • the piezoelectric elements 10 are electrically connected in series in order to provide together a signal which is twice the signal of a single piezoelectric element 10. In this manner, the sensitivity is increased without increasing the length of the elongate sections 11 and the support elements 8.
  • each support element 9 comprises a straight elongate silicone tube 13 of circular cross-section and a cylindrical elongate rod 14, which is rigid and also of circular cross-section.
  • Each such rod 14 comprises a straight section 14a, which extends over nearly the entire length or at least most of the length of the rod 14, and at one of its longitudinal ends an end section 14b bent by 90° as compared to the straight section 14a.
  • the rod 14 is dimensioned such that the rod 14 or, more particularly, the straight section 14a thereof can be selectively inserted into and removed from the tube 13 to selectively render the support element 9 rigid and flexible, respectively.
  • the length of the rod 14 and of the straight section 14a thereof are chosen to be greater than the length of the tube 13, to allow for rendering the support element 9 rigid along the entire length of the tube 13 while being able to easily remove the rod 14 when flexibility of the support element 9 is desired.
  • the diameter of the rod 14 is chosen such that insertion and removal are possible, but sufficient rigidity in the radial direction is also provided.
  • the bent end section 14b provides the advantage that the protrusion of the rod 14 above the base element 2 is limited, so that the entire arrangement is kept relatively flat, even if covered with a lid.
  • the rods 14 are straight along their entire length, i.e. that the bent section 14b is omitted.
  • a dish-shaped silicone element 15 having a circular shape and extending perpendicularly to the direction of extension of the tube 13 is attached.
  • the element 15 has rounded edges and, in the example illustrated, is identical to the elements 12. All elements 12 and 15 are arranged at the same distance from the base element 2.
  • the piezoelectric element 10 forms, together with electrical components not specifically shown, a piezoelectric transducer which is, in turn, electrically connected to the component 5.
  • the support elements 8 are arranged as close as possible to the component 5 in order to keep noise, interference and response time at a minimum.
  • the elements 12 and 15 constitute support portions which are suitable for attaching engineered heart tissue constructs thereto. More particularly, each pair of support elements 8 and 9 associated with the same cut-out 3 is adapted and intended for mounting an engineered heart tissue construct to the corresponding pair of support elements 12 and 15. If the support portions 12 and 15 are immersed into a liquid reconstitution mixture which is provided in a rectangular casting mold, the strip-shaped engineered heart tissue construct formed upon solidification directly attaches to the support elements 8 and 9 via the corresponding support portions 12 and 15. This is illustrated for an engineered heart tissue construct 16 for the rightmost pair of support elements 8 and 9. As illustrated, it is suspended in a slightly stretched state between the two support elements 8 and 9, wherein the tissue has attached to the support portions 12 and 15 at contact points 17. The dish-shaped support elements 12, 15 prevent that the engineered heart tissue construct slips off the support elements 8 and 9 upon culturing of the construct.
  • the support elements 8 constitute first support elements in accordance with the above general description
  • the support elements 9 constitute second support elements in accordance with the above general description
  • the elements 12 and 15 constitute corresponding support portions in accordance with the above general description.
  • Figure 2 shows in which manner the apparatus 1 may be advantageously used for measuring while two engineered heart tissue construct 16 mounted to the two pairs of support elements 8 and 9 are disposed inside two different wells 20 of a suitably dimensioned multi-well plate 21.
  • the dimensions of the multi-well plate 21 are adapted to the apparatus 1, such that the distance between the centers of the two cut-outs 3 is identical to the distance between the centers or longitudinal axes of two adjacent wells 20, that the distance between the two support elements 8, 9 associated with each cut-out 3 is smaller than the diameter of the wells 20, and that an engineered heart tissue construct 16 suspended on the elements 12 and 15 is spaced from the bottom of the well 20 if the base element 2 is located on the surface 22 of the multi-well plate 21 extending between the openings of the wells 20 and the two pairs of support elements 8 and 9 extend into adjacent ones of the wells 20.
  • the flexible strip 6 including electrical conduits can be conveniently guided around the multi-well plate 21 and comprises a connector 6a for connection to external devices.
  • the cut-outs 3 allow visual inspection of the engineered heart tissue constructs 16 without moving the apparatus 1.
  • the apparatus 1 may be covered during the measurement by a, preferably transparent, lid.
  • the apparatus 1 advantageously adds only little to the height of the multi-well plate 21.
  • the apparatus of the invention may be used in combination with casting molds to prepare engineered heart tissue constructs based on a liquid reconstitution mixture.
  • the apparatus can be arranged above the casting molds which contain the liquid reconstitution mixture such that the tips of the downwardly extending support elements are immersed in the reconstitution mixture.
  • Non-limiting examples for protocols for the preparation of engineered heart tissue constructs are provided below. Any other method which has been described in the art for the generation of engineered heart tissue based on liquid reconstitution mixtures can be used as well.
  • PROTOCOL 1 TISSUE CONSTRUCTS FROM RAT CARDIAC MYOCYTES
  • Unpurified heart cells were isolated from neonatal Wistar rats (postnatal day 0 to 3) by a fractionated DNase/Trypsin digestion protocol as previously described ( Zimmermann et al. (2000), Biotechnol Bioeng., 68:106-114 ). The resulting cell population was immediately processed to prepare the tissue constructs.
  • a reconstitution mix was prepared on ice as follows (final concentration): 4.1x10 6 cells/ml, 5 mg/ml bovine fibrinogen (stock solution: 200 mg/ml plus aprotinin 0.5 ⁇ g/mg fibrinogen in NaCl 0.9%, Sigma F4753), 100 ⁇ l/ml Matrigel (BD Bioscience 356235). 2xDMEM was added to match the volumes of fibrinogen and thrombin stock to ensure isotonic conditions. Casting molds were prepared by placing a teflon spacer in 24-well culture dishes and adding 1.6 ml 2% agarose in PBS (Invitrogen 15510-027) per well.
  • constructs were placed in a 37°C, 7% CO2 humidified cell culture incubator for 2 hours. 300 ⁇ l of cell culture medium was then added per well to ease removal of the constructs from agarose casting molds. The racks were transferred to new 24-well cell culture dishes. Constructs were maintained in 37°C, 7% CO 2 humidified cell culture incubator. Media was changed on Mondays, Wednesdays and Fridays.
  • the medium consisted of DMEM (Biochrom F0415), 10% horse serum (Gibco 26050), 2% chick embryo extract, 1% Penicillin/Streptomycin (Gibco 15140), insulin (10 ⁇ g/ml, Sigma-Aldrich 19278), tranexamic acid (400 ⁇ M, Sigma-Aldrich 857653) and aprotinin (33 ⁇ g/ml, Sigma Aldrich A1153).
  • the engineered heart tissue constructs contained evenly distributed amorphous round heart cells.
  • cells elongated, aligned along force lines, and started to beat as single cells at day 4 to 5.
  • Degradation and remodeling of extracellular matrix led to a marked reduction of construct size, increased cellular density, and formation of small groups of cardiac myocytes and, at day 7 to 9, to coherent beating.
  • force of contraction was sufficient to rhythmically deflect the posts. Measurements were routinely done between days 14 to 16.
  • cardiomyocytes appear as approximately 100 to 200 ⁇ um long spindle-shaped cells with maximum diameter of 10 to 20 ⁇ m.
  • PROTOCOL 2 TISSUE CONSTRUCTS FROM HUMAN EMBRYONIC STEM CELLS
  • HES2 cells HES2.R26, see Irion et al. (2007), Nature Biotechnology 25: 1477-82
  • Matrigel ® with CF1-MEF conditioned medium according to the protocol of Xu et al. (2001), Nature Biotechnology 19: 971-4 .
  • Confluent layers of hESC colonies were digested with collagenase IV (Gibco 17104, 1 mg/ml, 1 ml/10 cm 2 ) until edges of the colonies start to dislodge (10-20 minutes). Collagenase was removed and washed with 2 ml PBS/10 cm 2 .
  • CF1-MEF conditioned medium was added (1 ml/10 cm 2 ).
  • Embryoid bodies were generated by carefully scraping off colony fragments with a 5 ml-pipette tip. Colony fragments were collected and remaining colonies were detached with a cell scraper. EB formation was performed in ultra low attachment cell culture flasks (ULA-CCF, Corning 3815), with colony fragments of 2.5 cm 2 (undifferentiated hESC layer) per ml conditioned medium.
  • UAA-CCF ultra low attachment cell culture flasks
  • RPMI-B27 medium containing RPMI 1640 (Gibco 21875), B-27 supplement (2%, Gibco 0080085-SA), Penicillin/Streptomycin (0.5%, Gibco 15140) and HEPES (10 mM).
  • Y-27632 (10 mM, Biaffin PKI-27632-010) and growth factors were added.
  • EBs were collected in 50 ml tubes.
  • ULA-CCFs were washed twice with PBS, solutions were added to the tubes and centrifuged (4 minutes, 300 rpm).
  • Pelleted EBs were resuspended in 20 ml mesodermal induction medium (stage I, basic FGF (5 ng/ml, R&D 233-FB), Activin-A (6 ng/ml, R&D 338-AC), BMP-4 (10 ng/ml, R&D 314-BP), Y-27632 (10 mM)) and transferred back into the ULA-CCF. After 1-3 days EBs were collected in 50 ml tubes. ULA-CCFs were washed twice with PBS, and the solutions were added to the tubes and centrifuged (4 minutes, 300 rpm).
  • EBs were resuspendend in 15 ml cardiomyocyte induction medium (stage II, DKK-1 (150 ng/ml, R&D 5439-DK), VEGF (10 ng/ml, R&D 293-VE), Y-27632 (10 mM) and transferred back into the ULA-CCF. After 3 days EBs were collected in 50 ml tubes. ULA-CCFs were washed twice with PBS, solutions were added to the tubes and centrifuged (4 minutes, 300 rpm). EBs were resuspendend in 15 ml cardiomyocyte induction medium (stage III, DKK-1 (150 ng/ml), VEGF (10 ng/ml), basic FGF (5 ng/ml)).
  • EB formation and differentiation was performed at 95% humidity, 37°C, 5% oxygen, 5% CO2.
  • EBs were transferred to serum-containing medium (DMEM, Gibco 41965, 1% L-glutamine, 1% NEAA, 0.5% Penicillin/Streptomycin, 20% fetal bovine serum, 100 mM 2-mercaptoethanol) between day 12-15 and were kept at 20% oxygen. Onset of beating occurred between day 10 and day 15.
  • DMEM Gibco 41965, 1% L-glutamine, 1% NEAA, 0.5% Penicillin/Streptomycin, 20% fetal bovine serum, 100 mM 2-mercaptoethanol
  • EBs were dissociated with 500 ml of a pre-warmed mixture (37°C) of collagenase and trypsin (approximately 1 mg/ml collagenase in 0.5% trypsin solution) for 2-5 minutes under continuous trituration with a 100 ml pipette. Dissociated cells were washed twice by adding 1 ml of serum containing media and centrifugation for 2 min at 200 g.
  • EHTs were generated according to PROTOCOL 1 described above for neonatal rat cardiomyocytes. Specifically, 0.6 x10 6 differentiated cells were mixed with fibrinogen and thrombin and poured into slit-formed agarose casting molds in a standard 24-well plate in which two elastic silicone posts per well were inserted from above. After fibrin polymerisation (2 hours) silicone racks with 4 pairs of silicone posts each and the respective cell-fibrin gel were transferred to new 24 well plates and maintained under standard cell culture conditions (37°C, 95% humidity, 40% oxygen). Medium was changed on Mondays, Wednesdays and Fridays.
  • DMEM Biochrom F0415
  • 10% horse serum Gibco 26050
  • 2% chick embryo extract 1%
  • penicillin/streptomycin Gibco 15140
  • insulin 10 mg/ml, Sigma-Aldrich 19278
  • tranexamic acid 400 ⁇ M, Sigma-Aldrich 857653
  • aprotinin 33 mg/ml, Sigma-Aldrich A1153
  • Y-27632 (10 mM was added to the medium for the first 24 hours. EHTs were subjected to contraction analysis after 5 weeks, thus matching the age of 7-8 week old EBs.
EP13160816.8A 2013-03-25 2013-03-25 Verfahren und Vorrichtung zur Messung von Kontraktionseigenschaften von künstlich hergestellten Herzgewebe-Konstrukten Withdrawn EP2784149A1 (de)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP13160816.8A EP2784149A1 (de) 2013-03-25 2013-03-25 Verfahren und Vorrichtung zur Messung von Kontraktionseigenschaften von künstlich hergestellten Herzgewebe-Konstrukten
ES14722997T ES2881848T3 (es) 2013-03-25 2014-03-25 Procedimiento y aparato para medir continuamente las características de contracción de construcciones de tejido cardíaco diseñadas por ingeniería
PCT/EP2014/055981 WO2014154704A1 (en) 2013-03-25 2014-03-25 Method and apparatus for measuring contraction characteristics of engineered heart tissue constructs
EP14722997.5A EP2978835B1 (de) 2013-03-25 2014-03-25 Verfahren und vorrichtung zur kontinuierlichen messung von kontraktionseigenschaften von künstlich hergestellten herzgewebe-konstrukten
US14/780,199 US10172712B2 (en) 2013-03-25 2014-03-25 Method and apparatus for measuring contraction characteristics of engineered heart tissue constructs

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP13160816.8A EP2784149A1 (de) 2013-03-25 2013-03-25 Verfahren und Vorrichtung zur Messung von Kontraktionseigenschaften von künstlich hergestellten Herzgewebe-Konstrukten

Publications (1)

Publication Number Publication Date
EP2784149A1 true EP2784149A1 (de) 2014-10-01

Family

ID=48142616

Family Applications (2)

Application Number Title Priority Date Filing Date
EP13160816.8A Withdrawn EP2784149A1 (de) 2013-03-25 2013-03-25 Verfahren und Vorrichtung zur Messung von Kontraktionseigenschaften von künstlich hergestellten Herzgewebe-Konstrukten
EP14722997.5A Active EP2978835B1 (de) 2013-03-25 2014-03-25 Verfahren und vorrichtung zur kontinuierlichen messung von kontraktionseigenschaften von künstlich hergestellten herzgewebe-konstrukten

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP14722997.5A Active EP2978835B1 (de) 2013-03-25 2014-03-25 Verfahren und vorrichtung zur kontinuierlichen messung von kontraktionseigenschaften von künstlich hergestellten herzgewebe-konstrukten

Country Status (4)

Country Link
US (1) US10172712B2 (de)
EP (2) EP2784149A1 (de)
ES (1) ES2881848T3 (de)
WO (1) WO2014154704A1 (de)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3575392A4 (de) * 2017-01-26 2020-08-26 Osaka University Medium zur induzierung der differenzierung von stammzellen in mesodermale zellen und verfahren zur herstellung mesodermaler zellen

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106859683B (zh) * 2015-12-10 2020-08-04 上海联影医疗科技有限公司 心脏动态模拟装置
US11331027B2 (en) 2016-03-11 2022-05-17 University Of Washington System for magnetic detection of myocardial forces
DE102016110328B3 (de) 2016-06-03 2017-09-07 Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts, Universitätsmedizin Lochplatte
JP6793918B2 (ja) * 2016-09-02 2020-12-02 日本光電工業株式会社 測定装置

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030091979A1 (en) * 2000-01-27 2003-05-15 Thomas Eschenhagen Three-dimensional matrix body, and device and method for measuring contractions of a cell tissue
US20060105357A1 (en) * 1998-02-18 2006-05-18 Myomics, Inc. Tissue sensor and uses thereof
WO2006102890A1 (en) * 2005-03-29 2006-10-05 Danish Myo Technology A/S Tissue bath system with gas heating
EP1783206A1 (de) * 2005-11-08 2007-05-09 Universitätsklinikum Hamburg-Eppendorf Polyzyklisches künstliches Herzgewebe
EP1920789A1 (de) * 2006-11-11 2008-05-14 Universitätsklinikum Hamburg-Eppendorf Beutelartige Konstruktion mit Gewebe von Säugern

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060105357A1 (en) * 1998-02-18 2006-05-18 Myomics, Inc. Tissue sensor and uses thereof
US20030091979A1 (en) * 2000-01-27 2003-05-15 Thomas Eschenhagen Three-dimensional matrix body, and device and method for measuring contractions of a cell tissue
WO2006102890A1 (en) * 2005-03-29 2006-10-05 Danish Myo Technology A/S Tissue bath system with gas heating
EP1783206A1 (de) * 2005-11-08 2007-05-09 Universitätsklinikum Hamburg-Eppendorf Polyzyklisches künstliches Herzgewebe
EP1920789A1 (de) * 2006-11-11 2008-05-14 Universitätsklinikum Hamburg-Eppendorf Beutelartige Konstruktion mit Gewebe von Säugern

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3575392A4 (de) * 2017-01-26 2020-08-26 Osaka University Medium zur induzierung der differenzierung von stammzellen in mesodermale zellen und verfahren zur herstellung mesodermaler zellen

Also Published As

Publication number Publication date
WO2014154704A1 (en) 2014-10-02
ES2881848T3 (es) 2021-11-30
EP2978835A1 (de) 2016-02-03
US10172712B2 (en) 2019-01-08
EP2978835B1 (de) 2021-05-05
US20160030177A1 (en) 2016-02-04

Similar Documents

Publication Publication Date Title
EP2978835B1 (de) Verfahren und vorrichtung zur kontinuierlichen messung von kontraktionseigenschaften von künstlich hergestellten herzgewebe-konstrukten
Jackman et al. Engineered cardiac tissue patch maintains structural and electrical properties after epicardial implantation
Wang et al. Biowire model of interstitial and focal cardiac fibrosis
Hansen et al. Development of a drug screening platform based on engineered heart tissue
Li et al. Age-dependent functional crosstalk between cardiac fibroblasts and cardiomyocytes in a 3D engineered cardiac tissue
Gouveia et al. Flexible nanofilms coated with aligned piezoelectric microfibers preserve the contractility of cardiomyocytes
Bielawski et al. Real-time force and frequency analysis of engineered human heart tissue derived from induced pluripotent stem cells using magnetic sensing
Valls-Margarit et al. Engineered macroscale cardiac constructs elicit human myocardial tissue-like functionality
Powell et al. Mechanical stimulation improves tissue-engineered human skeletal muscle
Tang et al. How far cardiac cells can see each other mechanically
Liau et al. Functional cardiac tissue engineering
US10034738B2 (en) Cardiac tissue constructs and methods of fabrication thereof
US20130046134A1 (en) Methods of generating engineered innervated tissue and uses thereof
Cho et al. Reconstructing the heart using iPSCs: Engineering strategies and applications
Massai et al. Bioreactor platform for biomimetic culture and in situ monitoring of the mechanical response of in vitro engineered models of cardiac tissue
Jayne et al. Direct laser writing for cardiac tissue engineering: a microfluidic heart on a chip with integrated transducers
Biendarra-Tiegs et al. Human induced pluripotent stem cell-derived non-cardiomyocytes modulate cardiac electrophysiological maturation through connexin 43-mediated cell-cell interactions
WO2016036532A1 (en) Automated, multifunctional, engineered cardiac tissue culture and testing bioreactor system
Dou et al. Microengineered platforms for characterizing the contractile function of in vitro cardiac models
US11407969B2 (en) System and method for generating biological tissue
Tadevosyan et al. Engineering and assessing cardiac tissue complexity
Gaffney et al. Potential synergistic effects of stem cells and extracellular matrix scaffolds
Tao et al. Bioengineering cardiac tissue constructs with adult rat cardiomyocytes
US20190365951A1 (en) Human cardiac tissue construct, related methods and uses
Gowran et al. Young at heart: Pioneering approaches to model nonischaemic cardiomyopathy with induced pluripotent stem cells

Legal Events

Date Code Title Description
17P Request for examination filed

Effective date: 20130325

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20150402